ALKALOID PRODUCTION I N CA THARA,VTHUS ROSE US CELL CULTURES. T. ALKALOIDS FROM THE 1'7BG, 299Y, 340Y A N D 95lG CELL LINES' JAMES P. K U T X E YLEWIS , S.L. CHOI,P A W E KOLODZIEJCZTK, L STEPHEN K. SLEIGH, KEKSETHL. STUART and BRI.ASR . WORTH D e p a r t m e n t of Chemistry, C n i t ' e r s i t j of B r i t i s h C o l u m b i a , V e n c o u r e r , B.C., Canade 1'6T 1 Y6 and R. G . R. KURZ,K. B. CHATSOK and F. COXSTABEL P r a i r i e Regional Laboratory, X n t i o n e l Research Council, S a s ka toon, Saskatchewan, Canada S7'X7 01I .9 ABsTR.ACT.-Pht cell cultures, 176G, 299Y, 340T and 95lG, from C . roseus gave t h e following alkaloids: yohimbine ( 5 ) , isositsirikine (6), horhammericine (7), horhammerinine (8), vindolinine (9), 19-epivindolinine (lo), ajmalicine (3), lochnericine (14). vallesiachotamine (a), strictosidine (12), strictosidine lactam (111, lochrierinine (15) and N,N-dimethyl-tryptamine (13).
I n recent publications (1-6) we have reported the production of a large number of indole alkaloids both in callus and suspension cultures derived from Catharaitllrus roseus. Among these cell cultures there have been cell lines that produce catharanthine (1, Iboga) and a variety of -4spidosperma and Corynanthk alkaloids. I n a recent publication Stockigt et. al. ('7) have also reported the production of cultures that synthesize the more biosynthetically complex indole alkaloids. It was interesting to note that Stockigt found, with a fen- minor differences, the same alkaloids that we had earlier isolated and fully characterized. I n our continued investigations for cell lines that produce catharanthine (1) and/or vindoline (2), n-e have been examining the differences found in alkaloid production from cell cultures that have been derived, by anther explants, from different C. roseus plants. By following this approach we hoped to propagate a cell line that could synthesize a single or, perhaps, t n o major components. A cell line that could produce predominantly a single chemo-therapeutic component would have considerable significance for the pharmaceutical industry. Here we describe some of the results so far obtained.
RESULTS A S D DISCCSSIOS The growing of each innoculum and its subsequent propagation in cell suspension was carried out as described earlier (2). When the culture was harvested, the water was removed by freeze drying and the residue was extracted. Separation of the alkaloids was achieved by the same chromatographic procedure used earlier (2).
A culture derived from one plant and coded 176G was propagated; the alkaloids isolated are shown in table 1. The two major components are ajmalicine (3) (13.57, of total alkaloid content) and lochnericine (14) (8.57,), though there are significant amounts of yohimbine ( 5 ) (0.9% j , isositsirikine (6) (0.75%), horhammericine ( 7 ) (l.S5%), horhammerinine (8) ( l . l % ) , vindolinine (9) (2.27,) and 19-epivindolinine (10) (2.0%). A second cell line was derived from a plant coded 2991'. The alkaloid content is shoan in table 2. I n this case only two major alkaloids could be isolated: strictosidine (12) (0.35 g, S3Y0 of total alkaloid content) and strictosidine lactam (11) (0.03 g, 6y0of total alkaloid content), It is not clear a t the moment whether 1NRCC #18655. 536
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K-utney et al.: Cell Cultures of C. roseus
5.37
TABLE 1. Alkaloids isolated from the l i 6 G cell line.
,
I
5 of I-ohimbine ( 5 ) .. . . . . . . . , . . . . . . , . Isositsirikine ( 6 ) ., , , . . . . . . . . . . , Horhammericine ( 7 ) . . . . . . . . . . . . . Horhammerinine ( 8 ) . . . . . . . . . . . . Vindolinine ( 9 ) .. . , , . . . . . . . . . . . . . 19-Epivindolinine (10).. . . . . . . . . . Ajmalicine (3). . . . . . . . . . . . . . . . . . Lochnericine (14).. . . . . . . . . . , . . . ,
2.5 2.0 5.0 3.0 6.0 5.5 25.0 16.0
Total alkaloid content (2iO mg)
yc of Dry cell
0.9 0.75 1.85 1.1 2.2 2.0 13.5 8.5
0.004
r t (62.1 gm) 0.003 0.008 0.005 0.009 0.009 0.04 0.026
OCOCH,
Me0,C
a; OH
7
Me 0
CH,OH
OH
8
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9
;o2Me
ti 10
602Me
9 H OGlu "H
11
::0
12
14
(11) is present in the culture or is formed from (12) during the isolation procedure. Among the other components isolated were ajmalicine (3) (16 mg, 4.0%), vallesiachotamine (4) (17 mg, 4.2%)) vindolinine (9) (.3 mg, O . l % ) , horhammericine (7) (4 mg, 0.9%), horhammerinine (8) ( 2 mg, 0.5%), lochnericine (14) (2.5 mg, 0.6%) and lochnerinine (15) (2 mg, 0.5%). It is of interest that although strictosidine (12), the precursor of the indole alkaloids, was formed in abundance, the cell enzyme system did not seem to be able to transform it into more elaborate alkaloids. TABLE 2. Alkaloids isolated from t h e 299Y cell line.
% of Total alkaloid
of Dry cell wt (229 g)
4.0
